Our lab is interested in how gene expression contributes to animal development. Towards this end, we have chosen to examine a model gene expression program that includes three gene sets (intermolt, early, and late) which are expressed sequentially in the larval and prepupal salivary glands of Drosophila melanogaster and whose regulation is denpendent on the steroid hormone ecdysterone. Genetic and developmental data are consistent with the hypothesis that sequential expression of these gene sets is accomplished at the transcriptional level through a cascading regulatory mechanism. The 2B5 "early" gene(s), when disrupted by mutations, prevents this cascase and, thus, plays a key role in this regulation. We have cloned most of the genomic DNA in this region (greater than 125 kb) with the intent to determine how the 2B5 product(s) functions at the molecular level. The number of relevant genes in the region will be enumerated by determining the number of transcription units with the appropriate developmental expression using sensitive hybridization protocols. With this transcriptional organization in mind, each gene will be tested for its ability to complement mutants that eliminate the puffing cascade. This will be done by injecting cloned wild-type DNA segments into mutant salivary gland nuclei or pre-blastoderm animals. The molecular mechanism(s) by which the product(s) act will be approached by using recombinant DNA techniques to synthesize sufficient amounts of the protein product(s) to produce specific antibody reagents. These will be used to determine the intracellular location of the 2B5 product(s) in developing salivary glands and animals. We are particularly interested in testing the idea that this product(s) acts directly at the member sites that comprise the gene sets controlled by the 2B5 product(s).